Genetic immunization with cationic lipids

ABSTRACT

A method for immunization using genetic material is disclosed. Compositions for genetic immunization comprising cationic lipids and polynucleotides are also disclosed. Methods for using genetic immunization to produce polyclonal and monoclonal antibodies are also disclosed. A method for epitope mapping is also disclosed.

CROSS-REFERENCE TO OTHER APPLICATIONS

This application is a continuation of U.S. application Ser. No.10/906,246, filed Feb. 10, 2005, which is a divisional application toU.S. application Ser. No. 08/450,555, filed May 25, 1995, now U.S. Pat.No. 6,890,554, which is a continuation of U.S. application Ser. No.08/069,720, filed Jun. 1, 1993, now abandoned, all of which areincorporated by reference in their entirety herein to the extent notinconsistent with the disclosure herewith.

BACKGROUND OF THE INVENTION

The present invention is in the field of immunology. In particular, thisinvention is directed to methods of immunization using compositionscomprising cationic lipids and polynucleotide molecules which code forimmunogens. This invention is also directed to methods for producingpolyclonal and monoclonal antibodies from genetically immunized animals.This invention is further directed to the use of genetic immunization tomap protein epitopes.

Traditional methods of immunization are achieved by injection of amixture of antibodies which immunoreact with an invading pathogen (i.e.,passive immunization), or by vaccination, which stimulates the immunesystem to produce pathogen-specific antibodies. Since foreign antibodiesare cleared by the recipient, passive immunity confers only temporaryprotection. Vaccination confers longer-lasting active immunity.

In order to be effective, vaccination must generate humoral and/orcell-mediated immunity which will prevent the development of diseaseupon subsequent exposure to the corresponding pathogen. The pertinentantigenic determinants must be presented to the immune system in amanner that mimics a natural infection. Conventional viral vaccines mayconsist of inactivated virulent strains, or live-attenuated strains (Oldet al., Principles of Gene Manipulation: An Introduction to GeneticEngineering, Blackwell Scientific Publications, 4th edition, 1989). Ageneral problem with using a vaccine consisting of a virus is that manyviruses (such as hepatitis B virus) have not been adapted to grow tohigh titre in tissue culture and thus, cannot be produced in sufficientquantity (Id.). In addition, the use of inactivated viruses present apotential danger of vaccine-related disease resulting fromreplication-competent virus may remain in the inoculum. Outbreaks offoot-and-mouth disease in Europe have been attributed to this cause(Id.). On the other hand, attenuated virus strains have the potential torevent to virulent phenotype upon replication in the vaccinee. Thisproblem has been reported to occur about once or twice in every millionpeople who receive live polio vaccine (Id.). Moreover, encephalitis canoccur following measles immunization with attenuated virus (Roit, I. M.Essential Immunology, Blackwell Scientific Publications, Sixth Ed.,1988). Another disadvantage of using attenuated strains is thedifficulty and expense of maintaining appropriate cold storagefacilities (Id.). A major disadvantage associated with the use of livevirus vaccines is that persons with congenital or acquiredimmunodeficiency risk severe infections. Such persons include childrenin developing countries who are often immunodeficient because ofmalnutrition and/or infection with viruses or parasites (Id., Old etal., supra).

As a result of recent advances in molecular biology and peptidesynthesis, it is possible to produce purified viral proteins orsynthetic peptides for use in immunoprophylaxis (Murphy et al.,“Immunization Against Viruses,” in Virology, Fields et al., Eds., RavenPress, New York, pp. 349-370, 1985). Purified antigens may be producedby synthesizing peptides which represent immunologically importantdomains of surface antigens of the pathogen. The synthetic peptideapproach has been successfully used with an antigen determinant of thefoot and mouth disease virus (Id.). One problem with this approach isthat the poor antigenicity of synthetic peptides has required the use ofFreund's adjuvant to enhance the immune response in experimental animals(Id.). Since Freund's adjuvant cannot be used in humans, an effectiveadjuvant for human use must be developed (Id.). In addition, a singleantigenic site may not be sufficient to induce resistant since largesurface antigens usually contain several distinct immunological domainsthat elicit a protective humoral and/or cell-mediated response (Bracialeet al., J. Exp. Med. 153:910-923 (1981); Wiley et al., Nature289:373-378 (1981)). There may also be difficulties in stimulating animmunologic response to epitopes that are formed by noncontiguous partsof the linear protein molecule (Murphy, et al., supra). There isevidence that the majority of protein determinants are discontinuous andinvolve amino acid residues that are far apart in the primary amino acidsequence, but are brought into close juxtaposition by peptide folding(Roit, supra).

The alternative approach to preparing proteins for vaccines involves theuse of cloned viral DNA inserted into a suitable vector to produce viralprotein in prokaryotic or eukaryotic cells (Aldovini et al., The NewVaccines, Technology Review, pp. 24-31, January 1992). This approach,also, has several limitations. For example, one must devise suitableconditions for the optimal production of the recombinant protein ofinterest by the recombinant host cells. The protein product must beisolated and purified from the culture system, and obtained insufficient quantities for use as a vaccine. Finally, it may be necessaryto perform post-translational modifications of the purified protein(such as glycosylation and/or cleavage of a fusion protein).

An alternative to producing the recombinant antigen in vitro is tointroduce nucleic acid sequences coding for the antigen into the cellsof the vaccine. In this way, the antigen is produced in vivo by thevaccinee's cells and provokes the immune response. Tang et al. (Nature356:152-154 (1992)) have shown that it is possible produce an immuneresponse to human growth hormone protein in mice by propelling goldmicroprojectiles coated with plasmids containing human growth hormonegenomic sequences. The resultant variability in the production ofantibody production was hypothesized to arise from the operation of themicroprojectile device, or the coating of the DNA onto themicroprojectiles.

More recently, Ulmer et al. (Science 259:1745-1749 (1993)) injected aplasmid carrying the gene for influenza A nucleoprotein into thequadriceps of mice. The mice produced nucleoprotein antibodies,indicating that the gene was expressed in murine cells. The mice alsoproduced nucleoprotein-specific cytotoxic T lymphocytes which wereeffective in protecting the mice from a subsequent challenge with aheterologous strain of influenza A virus. Similarly, Wang et al. (Proc.Natl. Acad. Sci. USA 90:4156-4160 (1993)) observed that theintramuscular injection of a human immunodeficiency virus (HIV) type 1envelope DNA construct in mice generated antigen-specific cellular andhumoral immune responses. In addition, splenic lymphocytes derived fromthe inoculated mice demonstrated HIV-envelope-specific proliferativeresponses. Thus, direct inoculation of DNA coding for pathogenicantigens can provide an alternative to the use of viruses, proteins, orpeptides.

One problem with using naked DNA for inoculation is the low efficiencyof cellular uptake. For example, the protocol of Wang et al., supra,requires the injection of 100 micrograms of the DNA construct biweeklyfor a total of four inoculations. As described herein, the use ofcationic lipids as a carrier for DNA constructs provides a moreefficient means of genetic immunization. According to the presentinvention, genetic immunization can be achieved with as little as 5micrograms of a DNA construct, which has been complexed with cationiclipid.

Liposomes have been used as carriers of genetic information in thetransfection of tissue culture cells. A fundamental problem ofliposome-mediated transfection with liposomes comprising neutral oranionic lipids is that such liposomes do not generally fuse with thetarget cell surface. Instead, the liposomes are taken up phagocytically,and the polynuelcotides are subsequently subjected to the degradativeenzymes of the lysosomal compartment (Straubinger et al., MethodsEnzymol. 101:512-527 (1983); Mannino et al., Biotechniques 6:682-690(1988)). Another problem with conventional liposome technology is thatthe aqueous space of typical liposomes may be too small to accommodatelarge macromolecules such as DNA or RNA. As a result, typical liposomeshave a low capturing efficiency (Felgner, “Cationic Liposome-MediatedTransfection with Lipofectin™ Reagent,” in Gene Transfer and ExpressionProtocols Vol. 7, Murray, E. J., Ed., Humana Press, New Jersey, pp.81-89 (1991)).

Liposomes comprising cationic lipids interact spontaneously and rapidlywith polyanions such as DNA and RNA, resulting in liposome/nucleic acidcomplexes that capture 100% of the polynucleotide (Felgner et a., Proc.Natl. Acad. Sci. U.S.A. 84:7413-7417 (1987); Felgner et al., Focus11:21-25 (1989)). Moreover, the polycationic complexes are taken up bythe anionic surface of tissue culture cells with an efficiency that isabout ten to one hundred times greater than negatively charged orneutral liposomes (Felgner, “Cationic Liposome-Medicated Transfectionwith Lipofectin™ Reagent,” in Gene Transfer and Expression ProtocolsVol. 7, Murray, E. J., Ed., Humana Press, New Jersey, pp. 81-89 (1991)).In addition, the polycationic complexes fuse with cell membranes,resulting in an intracellular delivery of polynucleotide that bypassesthe degradative enzymes of the lysosomal compartment (Duzgunes et al.,Biochemistry 28:9179-9184 (1989); Felgner et al., Nature 337:387-388(1989)).

Various formulations of cationic lipids have been used to transfectcells in vitro (WO 91/17424; WO 91/16024; U.S. Pat. No. 4,897,355; U.S.Pat. No. 4,946,787; U.S. Pat. No. 5,049,386; and U.S. Pat. No.5,208,036). Cationic lipids have also been used to introduce foreignpolynucleotides into frog and rat cells in vivo (Holt et al., Neuron4:203-214 (1990); Hazinski et al., Am. J. Respr. Cell. Mol. Biol.4:206-209 (1991)). Therefore, cationic lipids may be used, generally, aspharmaceutical carriers to provide biologically active substances (forexample, see WO 91/17424; WO 91/1604; and WO93/03709). Thus, cationicliposomes can provide an efficient carrier for the introduction offoreign polynucleotides into host cells for genetic immunization.

Various cationic lipids are well-known in the prior art. One well-knowncationic lipid is N-[1-(2,3-dioleoyloxy)propyl]-N,N,N-trimethylammoniumchloride (DOTMA). The structure of DOTMA is:

DOTMA, alone or in a 1:1 combination withdioleoylphosphatidylethanolamine (DOPE) can be formulated into liposomesusing standard techniques. Felgner et al. (Proc. Natl. Acad. Sci. U.S.A.84:7413-7417 (1987)) have shown that such liposomes provide efficientdelivery of nucleic acids to cultured cells. A DOTMA:DOPE (1:1)formulation is sold under the name LIPOFECTIN™ (GIBCO/BRL: LifeTechnologies, Inc., Gaithersburg, Md.). An other commercially availablecationic lipid is 1,2-bis(oleoyloxy)-3-3-(trimethylammonia)propane(DOTAP, which differs from DOTMA in that the oleoyl moieties are linkedvia ester bonds, not either bonds, to the propylamine. DOTAP is believedto be more readily degraded by target cells.

A related group of known compounds differ from DOTMA and DOTAP in thatone of the methyl groups of the trimethylammonium group is replaced by ahydroxyethyl group. Compounds of this type are similar to the RosenthalInhibitor of phospholipase A (Rosenthal et al., J. Biol. Chem.235:2202-2206 (1960), which has stearoyl esters linked to thepropylamine core. The dioleoyl analogs of the Rosenthal Inhibitor (RI)are commonly abbreviated as DORI-ether and DORI-ester, depending uponthe linkage of the fatty acid moieties to the propylamine core. Thehydroxy group can be used as a site for further functionalization, forexample, by esterification to carboxyspermine.

Another class of known compounds has been described by Behr et al.(Proc. Natl. Acad. Sci. USA 86:6982-6986 (1989); EPO Publication 0 394111), in which carboxyspermine has been conjugated to two types oflipids. The structure of 5-carboxylspermylglycine dioctadecylamide(DOGS) is:

The structure od dipalmitoylphosphatidylethanolamine5-carboxyspermylamide (DDPES) is:

Both DOGS and DPPES have been used to coat plasmids, forming a lipidaggregate that provides efficient transfection. The compounds areclaimed to be more efficient and less toxic than DOTMA for transfectionof certain cell lines. DOGS is available commercially as TRANSFECTAM™(Promega, Madison, Wis.)

A cationic cholesterol derivative (DC-CHol) has been synthesized andformulated into liposomes in combination with DOPE (Gao et al., Biochim.Biophys. Res. Comm. 179:280-285 (1991). The structure of this compoundis:

Liposomes formulated with DC-Chol provide more efficient transfectionand lower toxicity than DOTMA-containing liposomes for certain celllines.

Lipopolylysine is formed by conjugating polylysine to DOPE. Thiscompound has been reported to be especially effective for transfectionin the presence of serum (Zhon et al., Biochim. Biophys. Res. Comm.165:8-14 (1991)). Thus, lipopolylysine may be an effective carrier forimmunization.

In addition, Gebeyhu et al. (co-pending U.S. application Ser. No.07/937,508; filed Aug. 28, 1992) have developed novel cationic lipidsaccording to the general formula:

wherein R₁ and R₂ separately or together are C₁₋₂₃ alkyl or

alkyl or alkenyl,

Z₁ and Z₂ separately or together are H or unbranched alkyl C₁₋₆

X₁ is -(CH₂)_(n)Br, Cl, F or 1, n=0-6 or

X₂ is -(CH₂)_(n)NH₂ n=0-6 or

X₃ is -NH-(CH₂)_(m)-NH₂ m=2-6 or

X₄ is -NH-(CH₂)₃-NH-(CH₂)₄-NH₂ or

X₅ is -NH-(CH₂)₃-NH-(CH₂)₄-NH(CH₂)₃-NH2

X₆ is

where p is 2-5, Y is H or other groups attached by amide or alkyl aminogroup or

X₉ is a polyamine, e.g., polylysine, polyarginine, polybrene, histone orprotamine or

X₁₀ is a reported molecule, e.g., biotin, folic acid or PPD, or

X₁₁ is a polysaccharide or substituted polysaccharide, or

X₁₂ is a protein or

X₁₃ is an antibody or

X₁₄ is an amine or halide reactive group or

X₁₅ is -(CH₂)_(n)-SH where r is 0-6 or

X₁₆ is -(CH₂)₈-S—S-(CH₂)₁-NH₂ where s is 0-6 and t is 2-6.

These compounds are useful either alone, or in combination with otherlipid aggregate-forming components (such as DOPE or cholesterol) forformulation into liposomes or other lipid aggregates. Such aggregatesare cationic and able to complex with anionic macromolecules such as DNAor RNA.

SUMMARY OF THE INVENTION

The present invention is directed to a method for eliciting an immuneresponse in an animal, comprising the steps of: (A) mixing at least onecationic lipid with a polynucleotide, coding for an antigenicdeterminant, thereby forming a cationic lipid-polynucleotide complex;and (b) administering the lipid-polynucleotide complex to the animal.

The present invention is also directed to a method for generating activeimmunity against an infectious disease in an animal, comprising thesteps of: (a) mixing at least one cationic lipid with a polynucleotide,coding for an antigenic determinant of an organism which is thecausative agent of the infectious disease, thereby forming a cationiclipid-polynucleotide complex; and (b) administering thelipid-polynucleotide complex to the animal; whereby active immunity tothe infectious disease is generated.

The present invention is also directed to such a genetic immunizationmethod wherein the polynucleotide is an expression vector comprising aDNA sequence coding for an immunogen, wherein the transcription of theDNA sequence is under the control of a promoter.

The present invention is further directed to a genetic immunizationmethod wherein the polynucleotide is an RNA molecule which codes of ranimmunogen.

The present invention is further directed to a method for producingpolyclonal antibodies comprising the use of the genetic immunizationmethod described above, and further comprising the step of isolating thepolyclonal antibodies from the immunized animal.

The present invention is also directed to a method for producingmonoclonal antibodies comprising the steps of: (a) mixing at least onecationic lipid with a polynucleotide thereby forming alipid-polynucleotide complex, wherein the polynucleotide comprises a DNAsequence coding for an immunogen; (b) administering thelipid-polynucleotide complex to at least one mouse; (c) removingB-lymphocytes from the immunized mice; (d) fusing the B-lymphocytes fromthe immunized mice with myeloma cells, thereby producing hybridomas; (e)cloning the hybridomas; (f) selecting positive clones which produceanti-immunogen antibody; (f) culturing the anti-immunogenantibody-producing clones; and (h) isolating anti-immunogen antibodiesfrom the cultures.

The present invention is also directed to a method for mapping theepitopes of a protein molecule, comprising the steps of: (a) fragmentingDNA molecules coding for the protein in a random manner; (b) subcloningthe DNA fragments in an expression vector; (c) mixing at least onecationic lipid with each expression vector subclone, thereby forming acationic lipid-expression vector complex with each expression vectorsubclone; (d) administering the cationic lipid-expression vectorcomplexes to mice; and (e) determining which of the DNA fragments arecapable of generating the production of antibodies in the mice.

DEFINITIONS

In the description that follows, a number of terms used in recombinantDNA technology are utilized extensively. In order to provide a clear andconsistent understanding of the specification and claims, including thescope to be given such terms, the following definitions are provided.

Cloning vector. A plasmid or phage DNA or other DNA sequence which isable to replicate autonomously in a host cell, and which ischaracterized by one or a small number of restriction endonucleaserecognition sites at which such DNA sequences may be cut in adeterminable fashion without loss of an essential biological function ofthe vector, and into which a DNA fragment may be spliced in order tobring about its replication and cloning. The cloning vector may furthercontain a marker suitable for use in the identification of cellstransformed with the cloning vector. Markers, for example, providetetracycline resistance or ampicillin resistance.

Expression vector. A vector similar to a cloning vector but which iscapable of enhancing the expression of a gene which has been cloned intoit, after transformation into a host. The cloned gene is usually placedunder the control of (i.e., operably linked to) certain controlsequences such as promoter sequences. Promoter sequences may be eitherconstitutive or inducible.

Recombinant Host. In general, a recombinant host may be any prokaryoticor eukaryotic microorganism or cell which contains the desired clonedgenes on an expression vector or cloning vector. This term is also meantto include those microorganisms that have been genetically engineered tocontain the desired gene(s) in the chromosome or genome of thatorganism.

Recombinant vector. Any cloning vector or expression vector whichcontains the desired cloned gene(s).

Host. Any prokaryotic or eukaryotic microorganism or cell that is therecipient of a replicable expression vector or cloning vector. A “host,”as the term is used herein, also includes prokaryotic or eukaryoticmicroorganisms or cells that can be genetically engineered by well knowntechniques to contain desired gene(s) on its chromosome or genome. Forexamples of such hosts, see Maniatis et al., Molecular Cloning: ALaboratory Manual, Cold Spring Harbor Laboratory, Cold Spring Harbor,N.Y. (1982).

Promoter. A DNA sequence generally described as the 5N region of a gene,located proximal to the start codon. The transcription of an adjacentgene(s) is initiated at the promoter region. If a promoter is aninducible promoter, then the rate of transcription increases in responseto an inducing agent. In contrast, the rate of transcription is notregulated by an inducing agent if the promoter is a constitutivepromoter.

Gene. A DNA sequence that contains information needed for expressing apolypeptide or protein.

Structural gene. A DNA sequence that is transcribed into messenger RNA(mRNA) that is then translated into a sequence of amino acidscharacteristic of a specific polypeptide.

Expression. Expression is the process by which a polypeptide is producedfrom a structural gene. The process involves transcription of the geneinto mRNA and the translation of such mRNA into polypeptide(s).

Transfection. Transfection refers to the transformation of a host cellwith DNA. The recombinant host cell expresses protein which is encodedby the transfected DNA.

Epitope. The part of a non-immunoglobulin antigen to which the variableregion of an antibody binds.

Antigenic Determinant. A protein or peptide which contains one or moreepitopes.

Immunogen. A protein or peptide which is capable of eliciting an immuneresponse due to the presence of one or more epitopes.

DETAILED DESCRIPTION OF THE INVENTION

The present invention is directed to a method for eliciting an immuneresponse in an animal by administering a cationic lipid-polynucleotidecomplex, wherein the polynucleotide codes for an antigenic determinant.

The present invention is also directed to a method for generating activeimmunity against an infectious disease in an animal by administering acationic lipid-polynucleotide complex, wherein the polynucleotide codesfor an antigenic determinant of an organism which is the causative agentof the infectious disease.

The present invention is also directed to such a genetic immunizationmethod wherein the polynucleotide is an expression vector comprising aDNA sequence coding for an immunogen, wherein the transcription of theDNA sequence is under the control of a promoter.

The present invention is further directed to a genetic immunizationmethod wherein the polynucleotide is an RNA molecule which codes for animmunogen.

The present invention is further directed to a method for producingpolyclonal antibodies comprising the use of the genetic immunizationmethod described above, and further comprising the step of isolating thepolyclonal antibodies from the immunized animal.

The present invention is also directed to a method for producingmonoclonal antibodies using B-lymphocytes from mice following geneticimmunization.

The present invention is also directed to a method for epitope mappingusing genetic immunization.

I. Cationic Liposomes

Any of the cationic lipids known in the prior art may be employed in thepractice of the claimed invention. See, for example, Felgner et al.(Proc. Natl. Acad. Sci. U.S.A. 84:7413-7417 (1987)); Felgner et al.(Focus 11:21-25 (1989)); Felgner (“Cationic Liposome-MediatedTransfection with Lipofectin™ Reagent,” in Gene Transfer and ExpressionProtocols Vol. 7, Murray, E. J., Ed. Humana Press, New Jersey, pp 81-89(1991)); WO 91/17424; WO 91/16024; U.S. Pat. No. 4,897,355; U.S. Pat.No. 4,946,787; U.S. Pat. No. 5,049,386; U.S. Pat. No. 5,208,036; Behr etal. (Proc. Natl. Acad. Sci. USA 86:6982-6986 (1989); EPO Publication 0394 111); Gao et al. (Biochim. Biophys. Res. Comm. 179:280-285 (1991));Zhou et al., (Biochim. Biophys. Res. Comm. 165:8-14 (1991)); andGebeychu et al. (co-owned U.S. application Ser. No. 07/937,508; filedAug. 28, 1992), the contents of which are fully incorporated byreference.

Preferred cationic lipids includeN-[1-(2,3-dioleoyloxy)propyl]-N,N,N-trimethylammonium chloride (DOTMA).The structure of DOTMA is:

DOTMA, alone or in a 1:1 combination withdioleoylphosphatiadylethanolamine (DOPE) can be formulated intoliposomes using standard techniques. A DOTMA:DOPE (1:1) formulation issold under the name LIPOFECTIN™ (GIBCO/BRL: Life Technologies, Inc.,Gaithersburg, Md.).

Another preferred commercially available cationic lipid is1,2-bis(oleoyloxy)-3-3-(trimethylammonia)propane (DOTAP), which differsfrom DOTMA in that the oleoyl moieties are linked via ester bonds, notether bonds, to the propylamine.

A related group of preferred cationic lipids differ from DOTMA and DOTAPin that one of the methyl groups of the trimethylammonium group isreplaced by a hydroxyethyl group. Compounds of this type are similar tothe Rosenthal Inhibitor of phospholipase A (Rosenthal et al., supra),which has stearoyl esters linked to the propylamine core. The dioleoylanalogs of the Rosenthal Inhibitor (RI) are commonly abbreviated asDORI-ether and DORI-ester, depending upon the linkage of the fatty acidmoieties to the propylamine core. The hydroxy group can be used as asite for further functionalization, for example, by esterification tocarboxyspermine.

In another class of preferred cationic lipids, carboxyspermine has beenconjugated to two types of lipids. The structure of5-caraboxylspermylglycine dioctadecylamide (DOGS) is:

The structure of dipalmitoylphosphatidylethanolamine5-carboxyspermylamide (DDPES) is:

DOGS is available commercially as TRANSFECTAM™ (Promega, Madison, Wis.).

Another preferred cationic lipid is a cholesterol derivative (DC-Chol)which has been synthesized and formulated into liposomes in combinationwith DOPE. The structure of this compound is:

Another preferred cationic lipid is lipopolylysine, which is formed byconjugating polylysine to DOPE.

Additional preferred cationic lipids are described by the generalformula:

wherein R₁ and R₂ separately or together are C₁₋₂₃ alkyl or

alkyl or alkenyl,

wherein q is 1 to 6,

Z₁ and Z₂ separately or together are H or unbranched alkyl C₁₋₆

X₁ is -(CH₂)_(n)Br, Cl, F or I, n=0-6 or

X₂ is -(CH₂)_(n)NH₂ n=0-6 or

X₃ is -NH-(CH₂)_(m)-NH₂ m=2-6 or

X₄ is -NH-(CH₂)₃-NH-(CH₂)₄-NH₂ or

X₅ is -NH-(CH₂)₃-NH-(CH₂)₄-NH(CH₂)₃-NH2

X₆ is

where p is 2-5, Y is H or other groups attached by amide or alkyl aminogroup or

X₉ is a polyamine, e.g., polylysine, polyarginine, polybrene, histone orprotamine or

X₁₀ is a reporter molecule, e.g., biotin, folic acid or PPD, or

X₁₁ is a polysaccharide or substituted polysaccharide, or

X₁₂ is a protein or

X₁₃ is an antibody or

X₁₄ is an amine or halide reactive group or

X₁₅ is -(CH₂)_(r)-SH where r is 0-6 or

X₁₆ is -(CH₂)_(s)-S—S-(CH₂)₁-NH₂ where s is 0-6 and t is 2-6.

These compounds are useful either alone, or in combination with otherlipid aggregate-forming components (such as DOPE or cholesterol) forformulation into liposomes or other lipid aggregates. Such aggregatesare cationic and able to complex with anionic macromolecules such as DNAor RNA.

II. Expression Vectors

One form of polynucleotide which can be used for genetic immunization isa plasmid expression vector for the expression of the immunogen proteinin eukaryotic cells. The eukaryotic expression vector comprises fourmain components. First, the plasmid must contain prokaryotic sequenceswhich code for a bacterial replication origin and an antibioticresistance marker. These prokaryotic sequences allow the propagation andselection of the plasmid within the bacterial host. Second, the plasmamust contain eukaryotic elements which control initiation oftranscription. These elements include promoter and, possibly, enhancersequences. Third, the plasmid must contain sequences involved in theprocessing of transcripts, such as polyadenylation sequences. Fourth,the plasmid must contain DNA sequences coding for the immunogen. TheseDNA sequences may be either genomic DNA sequences, or complementary DNA(cDNA) sequences. (For reviews of expression vectors, see Old et al.,supra, Sambrook et al., Molecular Cloning: A Laboratory Manual, 2ndEdition, Cold Spring Harbor Laboratory Press, 1989, Gorman, “HighEfficiency Gene Transfer into Mammalian Cells,” in DNA Cloning, VolumeII, Glover, D. M. Ed., IRL Press, Washington, D.C., pp. 143-190 (1985)).

DNA or cDNA molecules which encode an immunogen can be operably linkedinto the expression vector. Two DNA sequences (such as a promoter regionsequence and an immunogen encoding sequence) are said to be operablylinked if the nature of the linkage between the two DNA sequences doesnot (1) result in the introduction of a frame-shift mutation, (2)interfere with the ability of the promoter region sequence to direct thetranscription of the immunogen encoding gene sequence, or 3) interferewith the ability of the immunogen gene sequence to be transcribed by thepromoter region sequence.

A DNA sequence encoding an immunogen molecule may be recombined withvector DNA in accordance with conventional techniques, includingblunt-edged or stagger-ended termini for ligation, restricted digestionto provide appropriate termini, filling in of cohesive ends asappropriate, alkaline phosphatase treatment to avoid undesirablejoining, and ligation with appropriate ligases.

A wide variety of transcriptional and translational regulatory sequencesmay be employed, depending upon the nature of the host. For a mammalianhost, the transcriptional and translational regulatory signals may bederived from viral sources, such as adenovirus, bovine papilloma virus,simian virus, or the like, where the regulatory signals are associatedwith a particular gene which has a high level of expression. Inaddition, promoters from mammalian expression products, such as actin,collagen, myosin, etc., may be employed. Alternatively, a prokaryoticpromoter (such as the bacteriophage T3 RNA polymerase promoter) may beemployed, wherein the prokaryotic promoter is regulated by a eukaryoticpromoter (for example, see Zhou et al., Mol. Cell. Biol. 10:4529-4537(1990); Kaufman et al., Nucl. Acids Res. 19:4485-4490 (1991)).Transcriptional initiation regulatory signals may be selected whichallow for repression or activation, so that expression of the genes canbe modulated.

The expression of the desired immunogen molecule in animals requires theuse of eukaryotic regulatory regions. Such regions will, in general,include a promoter region sufficient to direct the initiation of RNAsynthesis. Preferred eukaryotic promoters include the promoter of themouse metallothionein I gene (Hamer et al., J. Mol. Appl. Gen.1:273-288(1982)); the TK promoter of Herpes virus (McKnight, S., Cell31:355-365 (1982)); the SV40 early promoter (Benoist et al., Nature(London) 290:304-310 (1981)); the Rous sarcoma virus promoter (Gorman etal., supra); and the cytomegalovirus promoter (Foecking et al., Gene45:101 (1980)).

As is widely known, translation of eukaryotic mRNA is initiated at thecodon which encodes the first methionine. For this reason, it ispreferable to ensure that the linkage between a eukaryotic promoter anda DNA sequence which encodes the desired immunogen molecule does notcontain any intervening codons which are capable of encoding amethionine (i.e., AUG). The present of such codons results either in theformulation of a fusion protein (if the AUG codon is in the same readingframe as the desired receptor molecule encoding DNA sequence) or aframe-shift mutation (if the AUG codon is not in the same reading frameas the desired receptor molecule encoding sequence).

The desired immunogen molecule encoding sequence and an operably linkedpromoter may be introduced into the cells of the vaccine either as anon-replicating DNA (or RNA) molecule, which may either be a linearmolecule or, more preferably, a closed covalent circular molecule. Sincesuch molecules are incapable of autonomous replication, the expressionof the desired receptor molecule may occur through the transientexpression of the introduced sequence. Alternatively, permanentexpression may occur through the integration of the introduced sequenceinto the host chromosome.

Preferably, the introduced sequence will be incorporated into a plasmidor viral vector capable of autonomous replication in the recipient host.Several possible vector systems are available for this purpose. Oneclass of vectors utilize DNA elements which provide autonomouslyreplicating extra-chromosomal plasmids, derived from animal viruses suchas bovine papilloma virus, polyoma virus, adenovirus, or SV40 virus. Asecond class of vectors relies upon the integration of the desired genesequences into the host chromosome. Additional elements may also beneeded for optimal synthesis of mRNA. These elements may include splicesignals, as well as transcription promoters, enhancers, and terminationsignals. The cDNA expression vectors incorporating such elements includethose described by Okayama, Mol. Cell. Biol. 3:280 (1983), and others.

Alternatively, the polynucleotide molecule can be an RNA molecule whichcodes for the desired immunogen. Sufficient quantities of such RNAmolecules may be obtained using in vitro transcription, followed by RNApurification. The technique of transcribing cloned DNA sequences invitro using DNA-dependent RNA polymerases is well-known in the art (forexample, see Sambrook et al., supra).

Any immunogen-encoding sequence can be used in this invention. Forexample, such immunogens include herpes simplex virus glycoprotein D,hepatitis B surface antigen, influenza virus haemagglutinin, and humanimmunodeficiency virus envelope antigen. In addition, the claimedinvention may be used to characterize the protein product of apolynucleotide sequence of unknown identify, as described below.

III. Use of the Lipid/Polynucleotide Complex

According to the present invention, the lipid-polynucleotide complex isused to carry out an in vivo transfection. Transfected cells express theprotein encoded by the polynucleotide, and may present the foreignprotein on the cell surface. As a result, the host animal mounts animmune response to the foreign protein, or immunogen.

Thus, the lipid/polynucleotide complex can be used as a vaccine toinduce active immunity. Preferably, such active immunity is induced inhumans, although the invention is not intended to be so limiting. Anyanimal which may experience the beneficial effects of the vaccines ofthe invention are within the scope of animals which may be treatedaccording to the claimed invention.

Genetic immunization may be performed by administering vaccinescomprising the cationic lipid and polynucleotide in a wide range ofdosages, and over a wide range of ratios. Effective dosages andformulations will depend upon a variety of factors (such as the speciesof the vaccinee), and can be determined by one of ordinary skill in theart. Illustrative dosages, formulations, and modes of administration areprovided below.

Cationic lipid-polynucleotide complexes are formed by mixing a cationiclipid solution with an equal volume of polynucleotide solution. Thecationic lipid and polynucleotides can be dissolved in any sterilephysiologically-compatible aqueous carrier. Preferably, cationic lipidand polynucleotides are dissolved in sterile saline (150 mM NaCl)., Thesolutions are mixed at ambient temperatures. Preferably, the solutionsare mixed at 25 degrees C. After mixing, the cationiclipid-polynucleotide complexes are incubated at room temperature,preferably for 15 to 45 minutes.

Administration of lipid/polynucleotide complexes of the presentinvention may be by parenteral, intravenous, intramuscular,subcutaneous, intranasal, or any other suitable means. The specificdosage administered may be dependent upon the age, weight, kind ofcurrent treatment, if any, and nature of the immunogen which will beexpressed. The initial dose may be followed by a booster dosage after aperiod of about four weeks to enhance the immunogenic response.

Since genetic immunization generates the production ofimmunogen-specific antibodies in the vaccine, the present invention isalso directed to methods of producing immunogen-specific antibodies.Polyclonal antibodies may be isolated and purified from vaccinatedanimals using procedures well-known in the art (for example, see Harlowet al., Antibodies: A Laboratory Manual, Cold Spring Harbor LaboratoryPress, 1988).

This invention is also directed to the use of genetic immunization toproduce monoclonal antibodies. According to this method, mice areinjected with a lipid/polynucleotide complex, and B-lymphocytes areisolated from the immunized mice. Monoclonal antibodies are producedfollowing the procedure of Kohler and Milstein (Nature 256:495-497(1975) (for example, see Harlow et al., supra). Briefly, monoclonalantibodies can be produced by immunizing mice with a cationiclipid-polynucleotide complex, verifying the presence of antibodyproduction by removing a serum sample, removing the spleen to obtainB-lymphocytes, fusing the B-lymphocytes with myeloma cells to producehybridomas, cloning the hybridomas, selecting positive clones whichproduce anti-immunogen antibody, culturing the anti-immunogenantibody-producing clones, and isolating anti-immunogen antibodies fromthe hybridoma cultures.

As an alternative to generating monoclonal antibodies to knownimmunogens, genetic immunization can be used to identify antigenicdeterminants in a protein by epitope mapping. According to this method,the polynucleotide of the lipid-polynucleotide complex codes for aportion of a protein molecule. Preferably, random fragments of the DNAencoding the complete protein molecule are generated using sonication(Deininger et al., Anal. Biochem. 129:216-223 (1983)) or partial DNase Idigestion (Anderson et al., Nucleic Acids Res. 9:3015-3027 (1981)), andencoded by blunt-end ligation into a suitable site of an expressionvector. Alternatively, DNA fragments for epitope mapping can be obtainedby treating DNA molecules with one ore more restriction endonucleases,or by using the polymerase chain reaction to synthesize DNA molecules.The generation of monoclonal antibodies by cells derived from theimmunized mice will indicate which segments of the protein molecule areimmunogenic.

In addition, the claimed invention may be used to characterize theprotein product encoded by a DNA or RNA sequence of known identity. Forexample, a genomic library can be constructed in a cosmid vector,wherein the expression of the cloned DNA fragments is regulated by apromoter. The genetic immunization technique can then be used toimmunologically characterize the protein products of the subclonedgenomic fragments.

As described above, genetic immunization protocols in which marked DNAis administered can require as much as 100 micrograms of a DNA constructper inoculation. In contrast, the use of cationic lipids as a carrierfor DNA constructs according to the claimed invention permits geneticimmunization with as little as 5 micrograms of a DNA construct. Thus,the claimed invention provides a more efficient means of geneticimmunization.

Having now generally described the invention, the same will be morereadily understood through reference to the following examples which areprovided by way of illustration, and are not intended to be limiting ofthe present invention, unless specified.

EXAMPLE 1 Evaluation of the Immunization Protocol

This series of experiments employed pSV2CAT plasmids which carry thebacterial chloramphenicol acetyl transferase (CAT) gene under thecontrol of the simian virus 40 promoter (SV; Gorman, “High EfficiencyGene Transfer into Mammalian Cells,” in DNA Cloning, Volume II, Glover,D. M. Ed., IRL Press, Washington, D.C., pp. 143-190 (1985)). Plasmid DNAwas isolated from bacterial cells by the alkaline lysis method, andpurified by isopyenic centrifugation in cesium chloride/ethidium bromidegradients (Maniatis et al., supra). These experiments employedLIPOFECTAMINE™ (BRL) as the cationic lipid. LIPOFECTAMINE™ is composedof2,3-dioleyloxy-N-[2(sperminecarboxamido)ethyl]-N,N-dimethyl-1-propanaminiumtrifluoroacetate (DOSPA), which is formulated with dioleoylphosphatidylethanolamine (DOPE) at a 3:1 (W:W) ratio in water. The cationic lipidsand plasmids were dissolved in sterile saline (150 mM NaCl).

Lipid/DNA complexes were administered to mice by intraperitoneally (IP)or intranasally (IN). For IN administration, lipid/DNA mixtures wereformed by mixing 25 microliters of a DNA solution with 25 microliters ofa lipid solution. For IP administration, lipid/DNA mixtures were formedby mixing 200 microliters of a DNA solution with 200 microliters of alipid solution. In these experiments, 5 micrograms of pSV2CAT were mixedwith 0, 15, 30, 45, or 60 micrograms of lipid. After mixing, thelipid/DNA complexes were allowed to sit at room temperature for 15-45minutes before administration. As a control, certain mice were injectedsubcutaneously (SC) with CAT protein (25 micrograms) in completeFreund's adjuvant.

A CAT enzyme-linked immunoassay (ELISA) was used to determine thepresence of CAT antibodies in mouse sera samples. Microwell titer plateswere prepared by incubating each well of the plate with 1 microgram/mlof CAT in 0.1 M sodium carbonate buffer, pH 9.5. The plates wereincubated for 18 hours at 4EC. The wells were blocked with 0.2%ovalbumin dissolved in phosphate-buffered saline (PBS) with 0.1%Tween020 (dilution buffer). Samples were diluted in the dilution buffer,and 200 microliters were added to a well on the plate. The plate wassealed, and then incubated for 60 minutes at 37 degrees C. Following awash, the plates were incubated with 0.1 micrograms/ml goat anti-mouseIgG—horseradish peroxidase conjugate. The plate was sealed and thenincubated for 30 minutes at 37 degrees C. Following a second wash, theplates were developed with 3,3′,5,5′-tetramethylbenzidine substrate atroom temperature. The reaction was stopped by adding 2N sulfuric acid.

Mice which were immunized with lipid/CAT protein developed an immuneresponse, although it was not as great as the respone generated byadministration of CAT protein in complete Freund's adjuvant. Severalmice injected IP with lipid/pSV2CAT generated an immune response whichwas weak, but clearly above background. Immunization by intranasaladministration gave results that varied among mice, but with a responsethat was greater than that seen with IP administration.

EXAMPLE 2 Comparison of Cationic Lipids as DNA Complexing Agents

These experiments compared the following cationic lipids:LIPOFECTAMINE™, LIPOFECTACE™ (BRL), DORI-ether (VICAL, Inc., San Diego,Calif.), DORI-ether/lysolipid (VICAL Inc., San Diego, Calif.), and abromo lipid (1-propanaminium,N-[2(2-bromo)ethyl]-N,N-dimethyl-2,3-bis(9-octadecenyloxy)-bromide).LIPOFECTACE™ is a 1:2.5 (W/W) liposome formulation ofdimethyldioctadecylammonium bromide (DDAB) anddioleoylphosphatidylethanolamine (DOPE). The bromo lipid was prepared asdescribed in co-pending U.S. application Ser. No. 07/937,508 (filed Aug.28, 1992), which is fully incorporated by reference.

Immunizations were performed by using IP administration of 5, 10, 20,40, or 80 micrograms of pSV2CAT, which was complexed with a cationiclipid in a 1:4 ratio (W/W; pSV2CAT/cationic lipid). One mouse receivedpSV2CAT only, one mouse received CAT protein only, and one mousereceived CAT protein in complete Freund's adjuvant. At about day 14,selected mice were given a booster shot.

The results were analyzed using Western Blot analysis. Nitrocellulosesheets were prepared by applying purified CAT protein to thenitrocellulose (0.1 micrograms CAT protein/cm nitrocellulose). Serasamples were diluted into dilution buffer and 2 ml were added to eachnitrocellulose strip. The strips were incubated for 90 minutes withrocking at room temperature. Following a wash, the strips were incubatedwith 0.1 micrograms/ml goat anti-mouse IgG-alkaline phosphataseconjugate. The strips were then incubated for 30 minutes at roomtemperature. The strips were developed with 1 ml of nitrobluetetrazolium/5-bromo-4-chloro-3-indoyl phosphate stable mix substrate.The strips were rinsed with distilled water following development anddried.

The results demonstrated that mice immunized with DNA and lipid producedanti-CAT antibodies. The booster shot did not seem to greatly enhancethe response. Western blot analysis showed that although the ELISAresults indicated different intensities of immune response, all theanimals injected with lipid-DNA complexes generated anti-CAT IgG.

EXAMPLE 3 Effect of Different Promoters on the Immune Response

These experiments used the bromo lipid (described in Example 2) orLIPOFECTACE™ as the cationic lipid. The CAT plasmids comprised eitherthe cytomegalovirus (CMV) promoter (Foecking et al., Gene 45:101(1980)), the Rous sarcoma virus (RSV) promoter (Gorman, “High EfficiencyGene Transfer into Mammalian Cells,” in DNA Cloning, Volume II, Glover,D. M., Ed., IRL Press, Washington, D.C., pp. 143-190 (1985)), or theSV40 promoter (described above). Mice were immunized IP, as describedabove, following the protocol: Lipid Lipid quantity DNA quantity BromoLipid or 20 micrograms  5 micrograms LIPOFECTACE ™ Bromo Lipid or 40micrograms 10 micrograms LIPOFECTACE ™ Bromo Lipid or 80 micrograms 20micrograms LIPOFECTACE ™

One set (i.e., two mice) were used to test each promoter. As controls,mice received DNA without lipid, CAT protein in complete Freund'sadjuvant, or no inoculation.

The results of ELISA analysis indicated that the SV40 promoter seemed towork the best followed by the CMV promoter and then, the RSV promoter.The bromo lipid gave more consistent results, compared withLIPOFECTACE™, in generating an immune response. The positive resultsobserved in the ELISA was confirmed by Western Blot analysis. In bothassay formats, the results obtained with DNA-lipid immunizations weregreater than the results observed with DNA alone.

Although the foregoing refers to particular preferred embodiments, itwill be understood that the present invention is not so limited. It willoccur to those of ordinary skill in the art that various modificationsmay be made to the disclosed embodiments and that such modifications areintended to be within the scope of the present invention, which isdefined by the following Claims.

All publications and patent applications mentioned in this specificationare indicative of the level of skill of those in the art to which theinvention pertains. All publications and patent applications are hereinincorporated by reference to the same extent as if each individualpublication or patent application was specifically and individuallyindicated to be incorporated by reference in their entirety.

1-11. (canceled)
 12. A method for mapping the epitopes of a protein molecule, comprising the steps of: (a) fragmenting DNA molecules coding for the protein molecule in a random manner; (b) subcloning the DNA fragments in an expression vector; (c) mixing at least one cationic lipid with each of the expression vector subclones, thereby forming a cationic lipid-expression vector complex with each of the expression vector subclone; (d) administering said cationic lipid-expression vector complex to an animal; and (c) determining which of the DNA fragments are capable of generating the production of antibodies in the animal.
 13. The method of claim 12, wherein a promoter of the expression vector is a SV40 promoter.
 14. The method of claim 12, wherein a promoter of the expression vector is an RSV promoter.
 15. The method of claim 7, wherein a promoter of the expression vector is a CMV promoter.
 16. The method of claim 12, wherein the cationic lipid is a formulation having a 3:1 ratio of 2,3-dioleoyl-N-[2(sperminecarboxyamido)ethyl]-N,N-dimethyl propanium trifluoroacetate and dioleoylphosphatidyl ethanolamine (DOPE).
 17. The method of claim 12, wherein the cationic lipid is a formulation having a 1:2.5 ration of dimethyldioctadecylammonium bromide and dioleoylphosphatidyl ethanolamine (DOPE).
 18. The method of claim 12, wherein the cationic lipid is a 1:1 formulation of N-(1-(2,3v-dioleoyloxy)propyl)-N,N,N-trimethylammonium chloride (DOTMA) and dioleoylphosphatidyl ethanolamine (DOPE).
 19. The method of claim 12, wherein the cationic lipid is 1-propanaminium, N-[2(2-bromo)ethyl]-N,N-dimethyl-2,3-bis(9-octadecenyloxy)bromide, N-(1-(2,3-dioleoyloxy)propyl)-N,N,N-trimethylammonium chloride (DOTMA), 1,2-bis(oleoyloxy)-3-3-(trimethylammonia)propane (DOTAP), 5-carboxyspermyglycine dioctadecylamide (DOGS) or dipalmitoylphosphatidylethanolamine 5-carboxyspermylamide (DPPES).
 20. The method of claim 12, wherein the cationic lipid is a DORI-ether or a lipopolylysine.
 21. The method of claim 12, wherein the cationic lipid is a cationic lipid having the formula:


22. The method of claim 12, wherein the cationic lipid has the formula:

wherein: R₁ and R₂, separately or together, are C₁₋₂₃ alkyl or —CO—C₁₋₂₃ alkyl or alkenyl; q is 1 to 6; Z₁ and Z₂, separately or together, are H or unbranched alkyl C₁₋₆ and X₁-X₁₆ are defined as follows: X₁ is -(CH₂)_(n)Br, Cl, F or I, where n=0-6; X₂ is -(CH₂)₂NH₂, where n=0-6; X₃ is -NH-(CH₂)_(m)-NH₂, where m=2-6; X₄ is -NH-(CH₂)₃-NH-(CH_(0.2))₄-NH₂; X₅ is -NH-(CH₂)₃-NH-(CH₂)₄-NH(CH₂)₃-NH₂; X₆ is

wherein p is 2-5, Y is H or an alkyl group attached by an amide or an alkyl amino group; X₉ is polylysine, polyarginine, polybrene, histone or protamine; X₁₀ is biotin, folic acid, -NHCO-fluorescein or PPD; X₁₁ is a polysaccharide or substituted polysaccharide; X₁₂ is a protein; X₁₃ is an antibody; X₁₄ is an amine; X₁₅ is -(CH₂)_(r)-SH, wherein r is 0-6; and X₁₆ is -(CH2)_(s)-S—S-(CH₂)_(t)-NH₂, wherein s is 0-6 and t is 2-6.
 23. The method according to claim 12, wherein the cationic lipid is further combined with dioleoylphosphatidyl ethanolamine (DOPE) or cholesterol.
 24. The method according to claim 12, wherein the cationic lipid is formulated into liposomes or other lipid aggregates.
 25. The method according to claim 12, wherein the DNA molecules are genomic DNA sequences.
 26. The method according to claim 12, wherein the DNA molecules are complementary DNA sequences.
 27. The method of claim 12, wherein said animal is a mouse.
 28. The method of claim 12, wherein said administering is intraperitoneal.
 29. The method of claim 12, wherein said administering is intranasal.
 30. The method of claim 12, wherein said administering is intramuscular.
 31. The method of claim 12, wherein the antibodies are monoclonal antibodies. 